EP2337633B1 - Dispositif permettant de mettre en uvre une pcr - Google Patents

Dispositif permettant de mettre en uvre une pcr Download PDF

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Publication number
EP2337633B1
EP2337633B1 EP10795211A EP10795211A EP2337633B1 EP 2337633 B1 EP2337633 B1 EP 2337633B1 EP 10795211 A EP10795211 A EP 10795211A EP 10795211 A EP10795211 A EP 10795211A EP 2337633 B1 EP2337633 B1 EP 2337633B1
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EP
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Prior art keywords
temperature
pcr
nucleic acid
sample
cavity
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EP10795211A
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German (de)
English (en)
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EP2337633A1 (fr
Inventor
Gerhard Hartwich
Norbert Persike
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Friz Biochem Gesellschaft fuer Bioanalytik mbH
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Friz Biochem Gesellschaft fuer Bioanalytik mbH
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
    • B01L7/5255—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones by moving sample containers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0803—Disc shape
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838—Capillaries
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks

Definitions

  • the present invention relates to a device for carrying out the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • PCR is a fundamental method for molecular biology that replicates DNA molecules and allows fast, sensitive direct detection of minute amounts of DNA or RNA. In recent years, this method has conquered the laboratories, as their application is very broad and complex. PCR has found its way into almost all areas of science and medicine, including forensic medicine, prenatal diagnostics, oncology and, not least, microbiological diagnostics. For example, PCR in the field of clinical diagnostics is usually the method of choice for z. B. detect pathogens. It is also used in the food industry as a detection method for harmful germs.
  • the PCR is an enzymatic reaction for the amplification of nucleic acid molecules, which essentially takes place in an aqueous or liquid reaction mixture with very small volumes.
  • the reaction mixture contains a nucleic acid-containing sample and the primers, nucleotides and a polymerase necessary for the reaction.
  • buffers and preferably bivalent ions such as B. Mg 2+ , the reaction mixture is adjusted so that prevail for the respective application optimum reaction conditions.
  • the polymerase chain reaction is based on a cycle of three steps occurring at different temperatures, namely denaturation, hybridization and extension.
  • the reaction mixture is heated to a temperature greater than 90 ° C, preferably 94 ° C to 95 ° C.
  • a temperature greater than 90 ° C preferably 94 ° C to 95 ° C.
  • the temperature is lowered to a so-called “annealing temperature”.
  • annealing temperature depends on the length and sequence of the primers and can be determined from these properties specifically for each primer. In general, the “annealing temperatures” in a temperature range of 55 ° C to 65 ° C, but application-specific can also be set lower or higher. The hybridization takes little time and takes place within seconds.
  • the temperature is further increased, preferably to 70 ° C to 74 ° C. This is the ideal working temperature for the usually used polymerases which, starting from the bound primers, grow further nucleotides on the resulting DNA strands. In the specified temperature range, loose connections between primers and those template DNA segments, which are not completely complementary, also break again.
  • the extension step is the step of the PCR, which usually involves the greatest amount of time.
  • the working or reaction rate of the polymerase is time-limited, that is, the shorter the optimal temperature of about 70 ° C to 74 ° C, the shorter the newly synthesized DNA strands remain.
  • several seconds (15 to 60 seconds) are sufficient in the extension step, but in Standard PCR method for very long DNA molecules to be synthesized a period of time in the range of minutes (from one to several minutes) chosen for DNA extension.
  • Each repetition of the above three steps doubles the number of copied DNA molecules. After 20 cycles, about one million molecules are formed from a single DNA double strand.
  • the number of cycles can be selected according to the type of application, the nature of the sample and the specific requirements and reaction conditions. Likewise, the setting of the time intervals for the individual steps is adapted to the specific requirements of certain types of applications.
  • RT-PCR reverse transcriptase PCR
  • an amplification is carried out with the aid of a reverse transcriptase starting from a sample RNA.
  • a hybrid double strand RNA / DNA is generated by the reverse transcriptase synthesizing a complementary to the present sample RNA DNA strand.
  • a "usual" PCR the resulting DNA can be used again as a template after denaturation of the hybrid.
  • the determination of the amount of DNA at the end of a PCR does not allow conclusions to be drawn directly for the number of molecules originally present, for example because of many reasons.
  • the conditions for the polymerases are not necessarily optimal and therefore the amplification does not proceed uniformly over the entire reaction time. Therefore, the quantification at the end of a PCR can be very inaccurate. Much more accurate quantification is possible if the number of DNA molecules formed during the reaction, so after every single cycle is detected. This quantification is usually done via a fluorescent label of the newly synthesized DNA molecules.
  • a quantification of the sample DNA is unavoidable, which is why a quantification option in real time is very much in demand in these areas.
  • the reaction mixture namely a nucleic acid-containing sample ("template DNA"), primer, nucleotides, polymerase and buffer are mixed in a reaction vessel, wherein usually between 10 .mu.l and 100 .mu.l reaction mixture are used in the reaction vessel.
  • the reaction vessel is fixed in a receiving unit and exposed to a number of temperature cycles.
  • the recording units are usually tempered metal blocks, which are equipped with recesses for receiving the reaction vessels.
  • the reaction vessels which have a capacity in the range of about 200 .mu.l to 500 .mu.l volume, are usually individual, sealable vessels or strips or plates with multiple wells, so-called multi-well strips or - plates. Number and relative distances of the wells of the multi-well plates or strips are matched to the wells of the temperature-controlled metal blocks, so that a fixation in the metal blocks is possible.
  • the temperature cycles are generated by a recurring heating and cooling of the temperature-controllable blocks, the control usually taking place via Peltier elements.
  • a major disadvantage of these repetitive heating and cooling phases is the time required for this.
  • the available devices have significant differences in heating and cooling rates of temperature-controlled blocks.
  • a rate of about 1 ° C to 10 ° C per second can be specified, the cooling rates are still slightly lower.
  • a time required between 15 seconds and 2 minutes is required, which results in 30 cycles for heating and cooling, a time requirement of up to one hour.
  • this expenditure of time represents a limitation of their efficiency.
  • WO 90/05023 a device for selectively setting the temperature of a sample to various values comprising a sample block having high thermal conductivity and a device for adjusting the temperature with at least two thermostattable bodies.
  • the sample receiving block is brought into contact with one of the bodies via a transport device.
  • the movement of the sample receiving block is in the form of a shift, so that at a defined time each sample is exposed to a certain temperature.
  • each sample can be brought into contact with different temperature zones sequentially by the sample vessel, namely the reaction chamber is moved stepwise by means of a stepping motor from one temperature zone to another temperature zone.
  • US 2002/0110899 A1 a "rotary thermocycler" with multiple stations for holding sample vessels, the stations being designed to set different temperatures.
  • the samples can be moved stepwise from one station to another with means for moving the sample tubes. This allows each sample to be sequentially exposed to the different temperatures.
  • the US Pat. No. 6,875,602 B2 discloses a portable thermal cycler in which a plurality of heating blocks are arranged on a rotating plate.
  • the sample containers may be in the form of capillary tubes and arranged in cassettes and are moved incrementally to the heating blocks.
  • the capillary tubes with the recorded samples are moved by the stepwise movement of the cassettes from one temperature zone to another temperature zone and thus exposed successively to the different temperatures.
  • the temperature control of the samples due to the transfer of the samples from one temperature to the next unit is faster than in conventional devices in which a repeated heating and cooling of a single temperature control unit is provided, but there is a constant problem that the heat transfer from the temperature control unit into the sample vessel and thus to the sample is not optimal. Due to the usually small contact surfaces between the sample vessel and tempering unit, for example in flat bottom vessels, which are placed on a temperature-controlled station, longer incubation times are necessary to ensure a temperature transfer to the sample, so that at any point of the sample volume, the desired temperature is reached and for the Duration of the desired reaction time prevails. Ultimately, therefore, the described devices do not lead to any noticeable time savings compared to conventional PCR devices.
  • the reaction mixture is moved through a channel that repeatedly passes through different temperature zones.
  • a PCR method and an apparatus for its implementation is described for example in EP 1 584 692 B1 disclosed.
  • disks are described on which concentric temperature zones z. B. be defined by means of two infrared ring heaters.
  • a channel passes through these temperature zones in a zig-zag shape.
  • the reaction mixture is moved by rotation of the disc by means of centrifugal force through the channel and thus passes through the different temperature zones.
  • a disadvantage of the in the EP 1 584 692 B1 described device is that the flow rate of the reaction mixture and thus the residence time in the individual temperature zones on the rotational speed of the disc is adjusted.
  • this flow rate can be very different depending on the viscosity of the sample used, whereby a meaningful and uniform thermal cycling for each sample would have to be redetermined.
  • the discs described also represent a very complex and therefore expensive consumable material.
  • a nucleic acid amplification device comprising a capillary tube reaction chamber, first and second heaters, and a positioning device.
  • the contacting of the sample with different temperature ranges takes place, inter alia, by "pumping" the sample through the capillary tube or by a movement of the heating device. Both a pumping of the sample through the capillary tube and a movement of the heater is cumbersome and requires a relatively high technical effort.
  • WO 2008/146754 A1 a device for performing a PCR method with a disc-shaped sample cell is known.
  • the sample cell has a cavity for receiving a sample.
  • the device comprises three independently adjustable temperature-controllable blocks, wherein in certain positions the cavity of the sample cell is in contact with two of the temperature-controllable blocks.
  • a disadvantage of the subject of WO 2008/146754 A1 are the relatively long periods of time required to adjust the temperature prevailing at the site of the sample.
  • US 2002/081669 A1 an apparatus for performing a PCR method with a disc-shaped sample cell.
  • the sample cell has a plurality of reaction chambers.
  • the device comprises three independently adjustable temperature-controlled blocks, wherein in certain positions a reaction chamber of the sample cell is in contact with two of the temperature-controllable blocks.
  • a disadvantage of the subject of US 2002/081669 A1 Again, these are the relatively long periods of time required to adjust the temperature prevailing at the site of the sample.
  • the present invention provides a device for carrying out a PCR method, wherein the device has at least one substantially in the form of a disc-containing sample cell having a cavity for receiving a sample.
  • the cavity provided for receiving the sample extends over a circular sector of the sample cell with a center angle of at least 180 °.
  • the device also comprises at least a first, a second and a third independently adjustable temperature control unit and at least one means for carrying out a rotational movement of the sample cell, wherein the three temperature control units define three spatially separate temperature zones.
  • the cavity provided for receiving the sample can be moved through the three temperature zones due to the rotational movement of the sample cell, wherein the cavity is in contact with at least two temperature zones, regardless of the position of the sample cell.
  • the means for performing a rotational movement is preferably an axis which is set in rotation by a corresponding drive.
  • the sample cell is preferably fixed on the axis such that the axis penetrates the sample cell in a substantially perpendicular direction. Most preferably, the axis penetrates the sample cell approximately in an area around its center.
  • rotationally symmetrical arrangement of the individual temperature control units about this rotatable axis the sample cell is moved evenly through the temperature zones by means of a rotary motion.
  • the simultaneous contact of the invention provided for receiving the sample cavity with at least two temperature zones has a positive effect on the effectiveness and speed of a PCR reaction.
  • Individual sections of the cavity, each of which receives a partial volume of the sample are each in contact with a temperature zone.
  • the temperature adjustment of these lower partial volumes to the temperatures necessary for carrying out the PCR reaction takes place very rapidly due to the reduced volume.
  • different temperatures can be set simultaneously in different cavity sections.
  • the reduction of the volume to be tempered and the parallelization of the heating and cooling steps for individual partial volumes lead to a time saving of up to two minutes per PCR cycle.
  • the cavity provided for receiving the sample is in contact with at least two temperature zones, independently of the position of the sample cell. This ensures that the PCR reaction is more effective and faster at each point in time and at each sample cell rotational position due to the positive effects mentioned above.
  • the sample cell has substantially the shape of a disk and the cavity provided for receiving the sample extends over a circular sector with a center angle of at least 180 °.
  • the cavity extends over a circular sector with a center angle of approximately 360 °.
  • preferred embodiments are those in which the cavity provided for receiving the sample extends over a circular sector with a center angle of approximately 225 ° or approximately 270 °. Due to the flat basic shape of the disc and arranged in the disc cavity, which, for example, via a machining production process such. B. milling is introduced into the disc, the surface / volume ratio is optimally designed for the fastest possible heat transfer.
  • the cavity provided for receiving the sample in the sample cell in the embodiments in which it extends over a circular sector with a center angle of approximately 360 ° substantially has the shape of a hollow cylinder.
  • the expression “essentially having the shape of a hollow cylinder” is to be understood that the shape of the cavity provided for receiving the sample deviates from an ideal hollow cylinder, since at least one, but usually two openings are provided for filling the sample.
  • the cavity thus has two ends and is not closed to an ideal hollow cylinder.
  • the cavity preferably takes the form of parts of a hollow cylinder.
  • the radii of the hollow cylinder are chosen so that the reaction volume necessary for a PCR can be absorbed by the hollow cylinder.
  • the hollow cylinder usually ends in two openings, which serve to fill the samples. When filling the sample through an opening, the air contained in the hollow cylinder can escape through the second opening and the sample is thereby evenly distributed. After filling, the openings can be closed in a liquid-tight manner by means of suitable stoppers or by laboratory fat and cyanoacrylate.
  • Particularly preferred embodiments are those in which a plurality of hollow cylinders are formed concentrically in the sample cell, or a plurality of parts of a hollow cylinder are arranged concentrically and / or on a circular line in the sample cell. This allows multiple PCR applications to be performed in parallel using a single sample cell.
  • the sample cell is formed in two parts in the form of a holding device for a capillary and a capillary for receiving the sample, wherein the capillary is connected in compression fit with the holding device.
  • the holding device has substantially the shape of a disc with an upper side, a lower side and a disc edge, and for receiving the capillary, a recess is provided on the upper side, on the underside or on the edge of the pane.
  • a circumferential recess since in this case offers the possibility, for example, a longer capillary or two or more shorter capillaries clamp in any arrangement, which in turn several PCR applications in parallel by means of a single sample cell can be performed.
  • the capillary is secured in the most favorable case in the press fit in the recess of the holding device, that only at most half the circumference of the capillary wall is received by the recess. This ensures that at least half the circumference of the capillary wall at the top, at the bottom or at the disk edge of the holding device with its surface or its edge terminate or beyond, thereby ensuring sufficient contact with the temperature control units and the associated optimal heat transfer ,
  • the capillaries connected to the holding device are made, for example, from polypropylene, from polycarbonate or from Teflon and can, for example, be tightly closed after being filled with the sample by being sealed at both ends over a small flame.
  • the first, the second and the third temperature control unit each have at least one temperature-controllable block.
  • the heatable blocks are made of a good thermal conductivity material, preferably made of metal, particularly preferably made of aluminum.
  • These blocks may be formed by means of a suitable component for energy transmission, preferably e.g. by means of a heating mat or a Peltier element and by means of a suitable device for temperature measurement, e.g. be brought to the desired temperatures by means of a platinum resistor with the aid of suitable control electronics (for example a PID controller).
  • the temperature zones defined by the tempering units can be expanded or expanded with suitable insulating materials.
  • suitable insulating materials for example, over one, over two or all three temperature-controllable blocks a larger-sized, open hollow body made of polystyrene foam (Styrofoam), be slipped, whereby a desired temperature in almost the entire hollow body interior can be adjusted by means of the temperature block and thus the temperature zone be extended can.
  • the individual temperature zones are isolated from each other.
  • styrofoam are suitable as insulating materials many known from the thermal insulation materials such as mineral fibers in the form of rock wool or glass wool, but also wood wool, hemp, felt or cork.
  • the temperature-controllable blocks of the first, the second and the third temperature control unit each have a notch for at least partially receiving the sample cell.
  • the indentations at least the edge regions of the sample cell are received by the heatable blocks in such a way that they are surrounded on three sides by the temperature-controllable block and the cavity with the sample is inserted between the blocks.
  • the cavity is arranged in the edge region of the sample cell such that the wall of the sample cell surrounding the cavity has a thickness of less than 500 ⁇ m both on the upper side of the preferably disk-shaped sample cell and on the underside and on the wafer edge. The sample is thus in close contact with the respective heatable block on three sides, which leads to a rapid temperature adaptation of the sample in the cavity.
  • the gap width of the notch can be adjusted by a suitable adjustment unit, such as e.g. Slotted holes varies and thereby the contact between sample cell and block can be optimally adjusted. This ensures a good heat transfer on the one hand, on the other hand, sample cells of different dimensions and geometries can be used. Additional introduction of lubricant, preferably mineral oil allows a low-friction movement of the sample cell and also leads to improved heat transfer.
  • a suitable adjustment unit such as e.g. Slotted holes varies and thereby the contact between sample cell and block can be optimally adjusted. This ensures a good heat transfer on the one hand, on the other hand, sample cells of different dimensions and geometries can be used. Additional introduction of lubricant, preferably mineral oil allows a low-friction movement of the sample cell and also leads to improved heat transfer.
  • the wall of the sample cell facing a tempering unit at least in the region of the cavity provided for receiving the sample, has a thickness of less than 500 ⁇ m, very particularly preferably less than 300 ⁇ m and particularly preferably less than 200 ⁇ m.
  • the thickness of the wall of the sample cell influences the rate of heat transfer, that is, the thinner the wall, the faster the heat is transferred from the tempering units to the sample.
  • the wall thickness can not be arbitrarily reduced for reasons of stability. Both heat transfer and stability are in turn dependent on the type of Therefore, the wall thickness is determined according to the material and geometry of the sample cell.
  • the sample cell has a diameter between 10 mm and 50 mm, more preferably a diameter between 20 mm and 30 mm.
  • the thickness of the sample cell is preferably between 0.2 mm and 1.5 mm, more preferably about 1 mm.
  • the cavity provided for receiving the sample preferably has a depth of 0.1 mm to 0.8 mm, particularly preferably a depth of 0.5 mm, and terminates in two openings.
  • the cavity 8 preferably has a volume between 1 .mu.l and 50 .mu.l, more preferably a volume of 20 .mu.l and can thus accommodate the required reaction volume of a PCR approach.
  • a sensor set up and designed for the detection of nucleic acid oligomer hybridization events in particular a sensor set up and designed for surface-sensitive detection of nucleic acid oligomer hybridization events, is provided in the cavity of the sample cell provided for receiving the sample.
  • the sensor consists essentially of a modified surface, wherein the modification consists in the connection of at least one type of probe nucleic acid oligomers.
  • the term "surface” refers to any support material that is capable of covalently or via other specific interactions binding derivatized or non-derivatized probe nucleic acid oligomers directly or after appropriate chemical modification.
  • the solid support may be made of conductive or non-conductive material. Methods for immobilizing nucleic acid oligomers on a surface are known to those skilled in the art.
  • it is a sensor designed and designed for spectroscopic, electrochemical or electrochemiluminescent detection of nucleic acid oligomer hybridization events.
  • a surface-sensitive detection enables the detection of exclusively bound to the surface signal nucleic acid oligomers.
  • the sensor is particularly preferably a DNA chip designed and designed for the electrochemical detection of nucleic acid oligomer hybridization events.
  • the modified surface has at least 2 spatially substantially separated regions, preferably at least 4 and in particular at least 12 spatially substantially separated regions.
  • the modified surface has at least 32, in particular at least 64, very particularly preferably at least 96 spatially substantially separated regions.
  • spatially substantially separated regions areas of the surface which are predominantly modified by attachment of a particular type of probe nucleic acid oligomer. Only in areas where two such spatially substantially separated regions are contiguous may a mixture of different types of probe nucleic acid oligomers occur.
  • a very particular advantage results from the fact that in addition a fourth temperature control unit is provided, wherein the fourth temperature control unit defines a fourth temperature zone which is spatially separated from the three temperature zones.
  • a fourth defined by a fourth temperature and spatially separated from the three temperature zones of the device temperature zone, it is possible to suspend portions of the sample cell, in particular the interior of the sample cell to a temperature that differs from the temperatures required to perform the PCR .
  • the geometry of the temperature-controllable block of the fourth temperature control unit and by choosing the arrangement in the device the area of the sample cell located in the fourth temperature zone can be determined.
  • the duration and frequency of incubation in the fourth temperature zone can be freely selected by adjusting the rotational speed of the sample cell.
  • the fourth temperature zone proves to be particularly advantageous for detection of nucleic acid oligomer hybridization events by means of a sensor.
  • the region of the sample cell in which the sensor is located is positioned in the fourth temperature zone, thereby providing optimum temperature conditions for detection.
  • a favorable temperature of about 50 ° C for this type of detection can be set.
  • the present invention also encompasses the use of the device according to the invention for carrying out a PCR, in particular for carrying out a real-time PCR.
  • an analysis method in an external detection system it is particularly advantageous to connect an analysis method in an external detection system to the PCR.
  • an external detection system for example, a miniaturized gel electrophoresis capillary comes into question.
  • a miniaturized gel electrophoresis capillary comes into question.
  • the wall of the sample cell in a region in which the cavity is formed preferably a circular predetermined breaking point.
  • the predetermined breaking point is adapted exactly to the diameter of the capillary, so that the wall of the sample cell is broken at this point by pressing the capillary to the predetermined breaking point, the capillary thereby liquid-tight can penetrate into the wall of the sample cell and the liquid in the cavity by capillary forces is pulled into the capillary.
  • the device according to the invention for carrying out a PCR can be used in a particularly advantageous manner in combination with a method for detecting nucleic acid oligomer hybridization events as an endpoint display and / or in real time.
  • the detection of the PCR products is carried out by a designed and designed for the detection of nucleic acid oligomer hybridization sensor, which is provided in the space provided for receiving the sample cavity of the sample cell.
  • Particularly preferred is one for surface-sensitive Detection of Sensor Established and Designed by Nucleic Acid Oligomer Hybridization Events.
  • the method for detecting nucleic acid oligomer hybridization events is particularly preferably an endpoint method for detecting the PCR products, comprising the steps of providing a modified surface, wherein the modification consists in the attachment of at least one type of probe nucleic acid oligomers, providing at least one species of signal nucleic acid oligomers wherein the signal nucleic acid oligomers are modified with at least one detection label and the signal nucleic acid oligomers have a complementary or substantially complementary portion to the probe nucleic acid oligomers, providing a sample with target nucleic acid oligomers, contacting a defined amount of the signal nucleic acid oligomers with the modified surface and contacting the sample and the target nucleic acid oligomers contained therein with the modified surface, detection of the signal nucleic acid oligomers and comparing the values obtained with the detection of the signal nucleic acid oligomers with reference values.
  • the signal nucleic acid oligomers in this case have a larger number of bases than the probe nucleic acid oligomers and have at least one docking section, wherein the docking section does not have a complementary or substantially complementary structure to a portion of the probe nucleic acid oligomers and wherein the target nucleic acid oligomers to the docking section have complementary or largely complementary section.
  • the docking section associates the target nucleic acid oligomers to the signal nucleic acid oligomers at a very high rate.
  • probe nucleic acid oligomers and signal nucleic acid oligomers are present upon addition of the target nucleic acid oligomers or probe nucleic acid oligomers and target nucleic acid oligomers upon addition of the signal nucleic acid oligomers as a hybridized duplex.
  • the added Nucleic acid oligomer component must be solved in accordance with the bonds of the hybridized duplex.
  • the two nucleic acid oligomer components have probe nucleic acid oligomers and signal nucleic acid oligomers according to the method described above a different number of bases.
  • the signal nucleic acid oligomers have a greater number of bases and provide a docking portion which is in a non-hybridized state because it does not have a complementary or substantially complementary structure to a portion of the probe nucleic acid oligomers.
  • the target nucleic acid oligomers now have a section which is complementary or largely complementary to the docking section.
  • the target nucleic acid oligomers can bind directly to this docking section without prior displacement of a hybridized component.
  • this displacement is due to the already done hybridization with the docking section at a much higher speed.
  • Another method for the detection of nucleic acid oligomer hybridization events comprises the steps of providing a modified surface, wherein the modification in the attachment at least one type of probe nucleic acid oligomer, providing a sample with target nucleic acid oligomers, providing a solution with at least one type of signal nucleic acid oligomers, wherein the signal nucleic acid oligomers are modified with at least one detection label, the signal nucleic acid oligomers one to the probe nucleic acid oligomers have complementary or largely complementary section and the signal nucleic acid oligomers have a complementary or substantially complementary to the target nucleic acid oligomers section, mixing the L solution with signal nucleic acid oligomers and the sample with target nucleic acid oligomers, contacting the mixture of signal nucleic acid oligomers and target nucleic acid oligomers with
  • a "largely complementary structure” is understood as meaning sequence segments in which a maximum of 20% of the base pairs form mismatches.
  • a "largely complementary structure” is preferably sequence sections in which a maximum of 15% of the base pairs form mismatches.
  • a "largely complementary structure” is a sequence segment in which a maximum of 10% of the base pairs form mismatches, and very particularly preferred are sequence segments in which a maximum of 5% of the base pairs form mismatches.
  • the detection of the signal nucleic acid oligomers is particularly preferably carried out by a surface-sensitive detection method, since in this case only the signal nucleic acid oligomers bound to the surface are detected.
  • a surface-sensitive detection method particularly preferred in this context are spectroscopic, electrochemical and electrochemiluminescent methods.
  • Particularly preferred as a spectroscopic method is detection of the fluorescence, in particular total internal reflection fluorescence (TIRF) of the signal nucleic acid oligomers.
  • cyclic voltammetry, amperometry, chronocoulometry, impedance measurement or scanning electrochemical microscopy are preferably used.
  • the present invention also encompasses a method for carrying out a PCR using the device according to the invention, wherein the Sample cell is moved by the spatially separated temperature zones by means of a rotary motion.
  • the rotational speed of the sample cell By selecting the rotational speed of the sample cell and matching it to the geometry of the temperature-controllable blocks, the residence time of the sample in the various temperature zones and thus the duration of the steps of a PCR cycle are determined. Since the different temperatures are already specified by the temperature control units, all parameters necessary for the PCR cycles can be set with a single parameter setting, namely the setting of the rotational speed of the sample cell. This leads to a user-friendly use of the device, as a complicated and time-consuming programming is eliminated.
  • the sample cell is moved at a constant speed.
  • Particularly preferred is a method for carrying out a PCR using the device according to the invention, in which the DNA chip is in the fourth temperature zone during the performance of an electrochemical detection of nucleic acid oligomer hybridization events.
  • the temperatures favorable for electrochemical detection with the aid of a DNA chip generally differ from the temperatures required for the PCR steps.
  • PCR and detection can take place at the respectively advantageous temperatures.
  • a particular advantage results from the use of the device according to the invention in one of the methods presented above in that PCR reaction and z.
  • the FIG. 1 shows a schematic representation of an embodiment of the device 1 according to the invention for carrying out a PCR.
  • the device 1 according to the invention comprises a first 2a, a second 2b and a third 2c independently adjustable temperature control unit, whereby three temperature zones, in the present example 96 ° C, 55 ° C and 72 ° C, are defined.
  • the three temperature control units 2a, 2b, 2c each have one temperable block 6 on.
  • the three blocks 6 are made of a good thermal conductivity material, preferably aluminum, but they can also consist of other suitable metal compounds.
  • the device 1 has a suitable means for energy transfer 13, preferably a heating mat or a Peltier element and a means for measuring temperature, preferably a platinum resistor or a digital thermometer.
  • a suitable control electronics eg Pl or PID controller
  • the three blocks 6 can be controlled exactly and thus the temperature zones are defined so that there are favorable temperatures for a PCR reaction.
  • the tempering units 2a, 2b, 2c are arranged on a base plate 10 so that they essentially describe the vertices of a triangle and their relative distance and / or their relative position to each other is freely adjustable. In principle, however, the tempering units can also be arranged in any other geometries. Centered between the temperature control units 2a, 2b, 2c is a means 3, 4 for carrying out rotational movement of the sample cell 5.
  • the means for performing a rotational movement comprises an axis 4 which is connected to the sample cell 5, and an electric motor 3.
  • the sample cell 5 of the illustrated example is, as exemplified by FIG. 3 is apparent, formed substantially disc-shaped, wherein the space provided for receiving the sample cavity 8 extends over a circular sector with a center angle of approximately 360 °.
  • Each block 6 of the three tempering units 2 a, 2 b, 2 c has a notch 7, which is designed for at least partially receiving the sample cell 5.
  • the sample cell 5 fixed to the axis 4 is partially inserted into each block 6, so that the sample cell 5 is arranged in one or more subareas in the three temperature zones and the cavity 8 is independent of the position of the sample cell 5 is in contact with three temperature zones.
  • the temperature control units 2a, 2b, 2c have a suitable adjustment unit 15, preferably oblong holes, over which the gap width of the notches 7 varies and can be adapted exactly to the thickness of the sample cell 5.
  • a suitable adjustment unit 15 preferably oblong holes, over which the gap width of the notches 7 varies and can be adapted exactly to the thickness of the sample cell 5.
  • the sample cell 5 is set in rotation, so that the individual portions of the sample cell 5 are guided through the three temperature zones.
  • a lubricant e.g., mineral oil
  • the introduction of a lubricant causes low friction rotation of the sample cell 5 as well as improved heat transfer.
  • the rotational speed or rotational frequency By varying the rotational speed or rotational frequency, the time in which the critical temperatures for the PCR reaction applied to the individual portions of the sample cell 5, can be varied.
  • FIG. 2a illustrated embodiment has a fourth temperature control unit 2d, whereby a fourth, spatially separated from the three temperature zones temperature zone is defined.
  • the fourth temperature control unit 2d may include a sensor 12 (see FIG FIG. 4 ), preferably hold a DNA chip at a certain temperature.
  • FIG. 2b A further embodiment of the device 1 with a plurality of indentations 7 in each temperature-controllable block 6 is shown in FIG. 2b shown. By means of this embodiment, several separate PCR reactions can be performed in parallel in one device.
  • the FIG. 3 shows a perspective view of an embodiment of a sample cell 5.
  • the sample cell 5 has a flat basic shape, preferably a cylindrical shape and consists of a plastic which has a temperature resistance up to at least 100 ° C. To observe the Sample behavior, it is advantageous if it is a transparent plastic.
  • the sample cell 5 preferably has a diameter d between 10 mm and 50 mm and a thickness b between 0.2 mm and 1.5 mm.
  • the cavity 8 for receiving the sample extends over a circular sector with a center angle of approximately 360 ° and has substantially the shape of a hollow cylinder with a preferred depth t of 0.1 mm to 0.8 mm, which ends in two openings 11 ,
  • the cavity 8 has a volume between 1 .mu.l and 50 .mu.l, preferably 20 .mu.l and can thus accommodate the required reaction volume of a PCR approach.
  • the sample cell 5 has a bore 9, by means of which the sample cell 5 in the device 1 according to the invention is attached to a means 3,4 for carrying out a rotational movement of the sample cell 5, preferably on a rotatable axis 4.
  • the openings 11 of the cavity 8 after filling with the PCR reaction mixture with laboratory grease, eg glisseal ® N and cyanoacrylate or by a suitable stopper 16, preferably made of rubber or plastic.
  • FIG. 4 shows a perspective view of another embodiment of the sample cell 5 according to the invention with an integrated sensor 12.
  • the cylindrical sample cell 5 has a cavity 8 for receiving the samples.
  • a bore 9 is provided, by means of which the sample cell 5 in the device according to the invention is attached to a means for carrying out a rotational movement of the sample cell, preferably on a rotatable axis.
  • the sensor 12 is in the present embodiment, a DNA chip.
  • the cavity 8, which has the shape of a hollow cylinder and contains the PCR reaction mixture, is widened by a branching channel 8.1, which communicates with the sensor surface.
  • a branching channel 8.1 which communicates with the sensor surface.
  • FIGS. 5a, 5b and 5c Further embodiments of a sample cell 5 with cavity 8, namely two-part embodiments are shown.
  • the sample cell 5 is designed as a holding device for a capillary, and the cavity 8 provided for receiving the sample is formed by a capillary.
  • the holding device 5 has for receiving the capillary 8 has a recess 18 into which the capillary 8 is clamped for clamping connection.
  • the FIG. 5a shows a plan view of a two-part embodiment during the FIG. 5b a vertical section through the in FIG. 5a represents a sample cell shown, in which a circumferential recess 18 for the clamped receiving the capillary 8 on the top of the disc-shaped holding device 5 extends.
  • the FIG. 5c shows a vertical section through a further two-part embodiment, in which a circumferential recess 18 for clamping-receiving the capillary 8 at the wafer edge of the holding device 5 extends.
  • the sample cell is constructed from two plastic parts, namely a lower part containing the cavity intended to receive the sample, and an upper part.
  • the bottom, cavity-containing plastic part of a sample cell having an approximate diameter of 15 mm and a thickness of about 1 mm is made of a polymethylmethacrylate blank, with an approximately 0.5 mm deep cavity 8 in Is milled into a hollow cylinder, which in two through openings 11 ends. In the center of the lower part of the sample cell, a bore 9 is introduced.
  • a piece of acrylic glass is glued to the lower plastic part with a suitable adhesive.
  • the acrylic glass sheet is thereby liquid-tightly connected to the lower, forming acrylic glass part.
  • an adhesive for acrylic glass for example, cyanoacrylate or dichloromethane can be used, for other polymers, some other adhesives may be necessary.
  • the openings 11 of the cavity 8 can be closed by laboratory grease (eg glisseal ® N) and cyanoacrylate or by a suitable rubber stopper.
  • materials with higher thermal conductivity can be used or the wall thicknesses of the materials can be reduced.
  • other methods of connecting the lower molding plastic part of the sample cell 5 to the upper plastic disk such as e.g. Welding process such as laser plastic welding conceivable.
  • Alternative cavity geometries with optionally incorporated sensors 12 can be produced in machining production processes, by forming processes (eg injection molding technology), by stereolithographic or other suitable methods.
  • An integrated sensor 12 may, for example, be contacted via an outgoing PCB circuit board 17, as in FIG FIG. 4 is shown.
  • a plurality of separate cavities 8 with a plurality of separate openings 11 can be introduced into a sample cell 5, as a result of which several PCR reactions in a sample cell 5 can take place in parallel. In this case too, it is possible to analyze the samples via a common or several sensors 12 (eg a sensor 12 per cavity 8).
  • a sample cell 5 enable a contamination-free "quasi-closed" liquid transfer from the cavity 8 of the sample cell 5 to an external detection system, for example a miniaturized gel electrophoresis capillary.
  • an external detection system for example a miniaturized gel electrophoresis capillary.
  • the wall of the sample cell in a region in which the cavity 8 is formed a substantially circular predetermined breaking point.
  • the predetermined breaking point can be introduced, for example, by tailor-made reduction of the wall thickness.
  • embodiments which additionally have a "female" connection side (inner cone) of a Luer-Lock connection at the predetermined breaking point.
  • a Luer-lock connection such as a syringe or cannula
  • the predetermined breaking point can be pierced and created a "quasi-closed" system between the cavity of the sample cell and the interior of the syringe.
  • PCR Polymerase Chain Reaction
  • a so-called master mix is prepared.
  • a reaction vessel e.g. A 2 ml micro screw cap tube labeled accordingly and placed in a cooling rack (0 ° C - 4 ° C).
  • the primers concentration mostly 10 ⁇ M
  • the dNTP mix c per dNTP 25 ⁇ M
  • dNTP deoxyribonucleoside triphosphates, i.e. DNA building blocks
  • standard PCR buffer and MgAc 100 mM
  • the DNA template to be amplified ie the isolated, purified and duplicated DNA material
  • the DNA template to be amplified is collected in 0.2 ml or 0.5 ml PCR reaction tubes, also PCR tubes (in the cooling rack at about 0 ° C - 4 ° C) submitted (usually 5 to 10 ul) and supplemented with Mastermix to usually 20 ul to 50 ul.
  • PCR tubes in the cooling rack at about 0 ° C - 4 ° C
  • Mastermix usually 20 ul to 50 ul.
  • water is introduced into a PCR tube instead of the DNA template (DNA-free) and filled with master mix to the corresponding final volume.
  • the polymerase is added to the provided PCR tubes (with template and master mix or with water and master mix in the case of the negative sample) and mixed by repeatedly aspirating and emptying the pipette.
  • the steps denaturation, annealing (primer hybridization) and elongation are repeated.
  • the double-stranded DNA templates are heated to 94-96 ° C to separate the strands.
  • the DNA is often heated for a long time (initialization) to ensure that both the starting DNA and the primer have completely separated and only single strands are present.
  • a temperature is set for about 30 seconds, which allows a specific attachment of the primer to the DNA. The exact temperature is determined by the length and the sequence of the primers (usually in the temperature range of about 55-65 ° C).
  • the DNA polymerase fills in the missing strands with free nucleotides.
  • the temperature depends on the working optimum of the DNA polymerase used (as a rule approx. 68 - 72 ° C). This step takes about 30 seconds per 500 base pairs, but varies depending on the DNA polymerase used.
  • a BioRad thermocycler (model iCycler) was used. 100 ⁇ l of common master mix was used for all reactions carried out, as listed in Table 1 below. 20 ⁇ l of template (DNA extract from Legionella pneumophila, about 100 DNA copies / 5 ⁇ l) were mixed with 80 ⁇ l of master mix mixed with Taq polymerase (BioTaq from BIOLINE) and in 4 batches of 25 ⁇ l each split (one for the PCR in the standard PCR thermocycler, three for the PCR using the device according to the invention). In addition, a negative control (5 ⁇ l water plus 20 ⁇ l master mix with polymerase, standard thermocycler) was used. All PCR conditions are given in Table 1.
  • Table 1 Mastermix Volume [ ⁇ l] Foreward Primer (10 ⁇ M) 6.30 Reverse primer (10 ⁇ M) 6.30 PCR Buffer * 21,00 MgAc (100mM) 2.63 DNTP mix (25 mM) 0.84 Taq polymerase (BioTaq) 0.53 H20 62.40 total 100.00 Parameters of the PCR using a standard thermocycler Temperature [° C] Time number of cycles 96 300 1 96 10 40 55 15 40 72 15 40 72 60 1 4 hold Parameters of the PCR using the device according to the invention A) Denaturation block Temperature [° C] Time cycles A 96 300 Rotation with approx.
  • the reaction time of the PCR of about 45 min (standard PCR) could be shortened to 25 min.
  • Optimized heater block geometries and minimized wall thicknesses of the sample cell should allow a further reduction of the reaction time for PCR to approximately 10 minutes or less.
  • the result of the PCR was checked by agarose gel electrophoresis.
  • Agarose gel electrophoresis is a method by which DNA fragments, in particular the PCR products, can be identified by their size.
  • the DNA is introduced into an agarose gel and then applied a voltage. As a result, shorter DNA fragments move faster towards the positive pole than longer DNA fragments.
  • the length of the PCR product can be determined by comparison with a DNA ladder containing DNA fragments of known size and co-running with the sample in the gel.
  • FIG. 6 Figure 4 shows a photograph of the gel electrophoresis analyzed PCR products obtained in the experiments summarized in Table 1.
  • A The gel electrophoretic analysis by means of standard PCR reaction PCR products obtained in a standard cycler designated A.
  • the negative control by standard PCR reaction performed in a standard cycler is designated B.
  • the gel electrophoretic analysis of the PCR products obtained using the device according to the invention are dependent on the rotation speed of the sample cell at C (0.5 revolutions per minute), D (1 revolute per minute) and E (2 revolutions per minute) designated.
  • F denotes a DNA ladder.
  • FIG. 6 clearly shows that the same expected approximately 150 bases long PCR product obtained by a standard PCR reaction in a standard cycler as when performing the PCR in a device according to the invention at three different rotational speeds of the sample cell.

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Claims (15)

  1. Dispositif (1) pour effectuer un procédé de PCR comprenant
    - au moins une cellule d'échantillon (5) présentant essentiellement la forme d'un disque, comprenant une cavité (8) pour recevoir un échantillon, sachant que la cavité (8) prévue pour recevoir l'échantillon s'étend sur un secteur circulaire de la cellule d'échantillon (5) avec un angle au centre d'au moins 180°,
    - au moins une première (2a), une deuxième (2b) et une troisième (2c) unités tempérées (2a, 2b, 2c) pouvant être réglées indépendamment l'une de l'autre, sachant que les trois unités tempérées (2a, 2b, 2c) définissent trois zones de température séparées physiquement l'une de l'autre et
    - au moins un moyen (3, 4) pour effectuer une rotation de la cellule d'échantillon (5), sachant que la cavité (8) prévue pour recevoir l'échantillon peut être déplacé du fait de la rotation de la cellule d'échantillon (5) à travers les trois zones de température et la cavité (8) est en contact avec au moins deux zones de température, indépendamment de la position de la cellule d'échantillon (5).
  2. Dispositif (1) selon la revendication 1, caractérisé en ce que la cavité (8) prévue pour recevoir l'échantillon s'étend sur un secteur circulaire de la cellule d'échantillon (5) avec un angle au centre s'approchant de 360°.
  3. Dispositif (1) selon la revendication 2, caractérisé en ce que la cavité (8) prévue pour recevoir l'échantillon dans la cellule d'échantillon (5) présente approximativement la forme d'un cylindre creux.
  4. Dispositif (1) selon l'une des revendications précédentes, caractérisé en ce que la cellule d'échantillon (5) est formée en deux parties sous la forme d'un dispositif de fixation pour un tube capillaire et sous la forme d'un tube capillaire pour recevoir l'échantillon, sachant que le tube capillaire est relié au dispositif de fixation en position serrée.
  5. Dispositif (1) selon la revendication 4, caractérisé en ce que le dispositif de fixation pour le tube capillaire est formé essentiellement sous forme d'un disque avec une face inférieure, une face supérieure et une bordure de disque, sachant que pour recevoir le tube capillaire, un évidement est prévu sur la surface supérieure et/ou sur la face inférieure et/ou sur la bordure de disque.
  6. Dispositif (1) selon l'une des revendications précédentes, caractérisé en ce que la première (2a), la deuxième (2b) et la troisième (2c) unités tempérées présentent respectivement au moins un bloc (6) pouvant être mis en température.
  7. Dispositif (1) selon la revendication 6, caractérisé en ce que les blocs (6) pouvant être mis en température de la première (2a), la deuxième (2b) et la troisième (2c) unités tempérées présentent respectivement une encoche (7) pour recevoir la cellule d'échantillon (5) au moins partiellement.
  8. Dispositif (1) selon l'une des revendications précédentes, caractérisé en ce que la paroi de la cellule d'échantillon (5) tournée vers une unité tempérée (2a, 2b, 2c) présente, au moins au niveau de la cavité prévue pour recevoir l'échantillon, une épaisseur inférieure à 500 µm.
  9. Dispositif (1) selon l'une des revendications précédentes, caractérisé en ce que dans la cavité (8) de la cellule d'échantillon (5) prévue pour recevoir l'échantillon, un capteur (12) installé et conçu pour la détection d'hybridations d'oligomères d'acide nucléique est prévu, en particulier un capteur (12) installé et conçu pour la détection sensible en surface d'hybridations d'oligomères d'acide nucléique.
  10. Dispositif selon la revendication 9, caractérisé en ce qu'il s'agit d'un capteur (12) installé et conçu pour la détection spectroscopique, électrochimique ou électrochimioluminescente d'hybridations d'oligomères d'acide nucléique.
  11. Dispositif selon la revendication 9, caractérisé en ce qu'il s'agit d'une puce à ADN (12) installée et conçue pour la détection électrochimique d'hybridations d'oligomères d'acide nucléique.
  12. Dispositif selon l'une des revendications précédentes, caractérisé en ce qu'une quatrième unité tempérée (2d) est prévue, sachant que la quatrième unité de tempérée (2d) définit une quatrième zone de température séparée physiquement des autres zones de température.
  13. Emploi d'un dispositif selon l'une des revendications 1 à 12 pour effectuer une PCR, en particulier pour effectuer une PCR en temps réel.
  14. Procédé pour effectuer une PCR en employant un dispositif selon l'une des revendications 1 à 12, caractérisé en ce que la cellule d'échantillon (5) est déplacée à vitesse constante à travers les zones de température séparées physiquement les unes des autres.
  15. Procédé pour effectuer une PCR en employant un dispositif selon la revendication 12, caractérisé en ce que la puce à ADN (12) se trouve dans la quatrième zone de température pendant qu'une détection électrochimique d'hybridations d'oligomères d'acide nucléique est effectuée.
EP10795211A 2009-11-05 2010-11-05 Dispositif permettant de mettre en uvre une pcr Not-in-force EP2337633B1 (fr)

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DE102009044431A DE102009044431A1 (de) 2009-11-05 2009-11-05 Vorrichtung zur Durchführung einer PCR
PCT/DE2010/075120 WO2011054353A1 (fr) 2009-11-05 2010-11-05 Dispositif permettant de mettre en oeuvre une pcr

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CA2837127C (fr) * 2011-05-24 2019-09-17 Ingeny PCR B.V. Systeme pour et procede de changement de temperatures de substances
KR101481054B1 (ko) 2011-11-15 2015-01-14 한국기계연구원 핵산 자동 분석 장치
DE102011056606B3 (de) 2011-12-19 2013-01-03 Friz Biochem Gesellschaft Für Bioanalytik Mbh Verfahren zur elektrochemischen Detektion von Nukleinsäureoligomer-Hybridisierungsereignissen
DE102016208972A1 (de) 2016-05-24 2017-11-30 Hahn-Schickard-Gesellschaft für angewandte Forschung e.V. Fluidikmodul, Vorrichtung und Verfahren zum biochemischen Prozessieren einer Flüssigkeit unter Verwendung von mehreren Temperaturzonen
DE102020106865A1 (de) 2020-03-12 2021-09-16 Analytik Jena Gmbh Anordnung und Verfahren zur PCR mit mehrkanaliger Fluoreszenzmessung für räumlich verteilte Proben

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US4902624A (en) * 1987-11-23 1990-02-20 Eastman Kodak Company Temperature cycling cuvette
DE8813773U1 (de) * 1988-11-03 1989-01-05 Max-Planck-Gesellschaft zur Förderung der Wissenschaften eV, 37073 Göttingen Gerät zum wahlweisen Einstellen der Temperatur einer Probe auf verschiedene Werte
DE69429038T2 (de) * 1993-07-28 2002-03-21 Pe Corp Ny Norwalk Vorrichtung und Verfahren zur Nukleinsäurevervielfältigung
CA2255850C (fr) * 1998-12-07 2000-10-17 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Appareil de thermocyclage rotatif
US6706519B1 (en) * 1999-06-22 2004-03-16 Tecan Trading Ag Devices and methods for the performance of miniaturized in vitro amplification assays
FR2812306B1 (fr) * 2000-07-28 2005-01-14 Gabriel Festoc Systeme d'amplification en chaine par polymerse de sequences nucleiques cibles
AU2003270832A1 (en) * 2002-09-24 2004-04-19 U.S. Government As Represented By The Secretary Of The Army Portable thermocycler
JP2005295877A (ja) 2004-04-09 2005-10-27 Taiyo Yuden Co Ltd 核酸分析方法、分析装置及び分析用ディスク
WO2008034896A2 (fr) * 2006-09-22 2008-03-27 Eppendorf Array Technologies S.A. Identification et quantification d'une pluralité de (micro)organismes biologiques ou de leurs composants
CN101802163A (zh) * 2007-05-23 2010-08-11 信诚医疗有限公司 反应液用容器，使用这种容器的反应促进装置，及其方法
US9399219B2 (en) * 2009-02-13 2016-07-26 Frank Leo Spangler Thermal Array

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DE102009044431A1 (de) 2011-06-22
WO2011054353A1 (fr) 2011-05-12
US20120088234A1 (en) 2012-04-12

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